The long-term goal of this program is to understand the molecular mechanisms by which different regulatory pathways interact to control expression of the Neurospora crassa arg-2 gene. This gene encodes the mitochondrially localized small subunit of arginine-specific carbamoyl phosphate synthetase. The major goal of this work is to define the intragenic and extragenic elements responsible for regulation of Arg-2 by Arg availability. arg-2 is regulated by at least three distinct mechanisms: (1)Arg-specific control negatively regulates expression in response to Arg. (2)Cross-pathway control positively regulates expression in response to limitation for a variety of amino acids, including Arg. (3) Developmental control regulates arg-2 expression in response to as yet unidentified developmental signals. Each of these pathways influences the level of arg-2 transcript. There is evidence for a translational component to Arg-specific control mediated through a 24 codon upstream open reading frame (uORF) in the transcript. Specific aims are as follows: 1. The contributions of transcriptional and translational components to regulation by Arg will be defined. The effects of existing mutations in arg-2 and at regulatory loci will be examined. 2. Novel mutations that affect gene expression in response to Arg will be obtained and characterized. Selection can be accomplished using cells harboring a recombinant gene in which E coli hph, which encodes hygromycin phosphotransferase, has been fused to arg-2 control sequences. These cells are resistant to the antibiotic hygromycin in medium lacking Arg and are sensitive to the antibiotic in medium containing Arg. 3. Mutations will be introduced in vitro into arg-2 regulatory sequences, and their effects on regulation will be analyzed in vivo by analyzing the expression of reporter genes - for example E coli lacZ - to which the sequences are fused. 4. The interactions of factors with arg-2 sequences will be characterized through in vitro DNA and RNA-binding studies as a complementary approach to in vivo studies. An understanding of how arg-2 expression is controlled is of practical as will as fundamental interest because N crassa is related to pathogenic fungi and to fungi used for the production of antibiotics.